The long-term objective of this project is to understand viral RNA processing at the molecular level. The current proposal focuses on mRNA 5' capping, which entails a series of reactions catalyzed by triphosphatase, guanylyltransferase, and methyltransferase enzymes. This application embraces three distinct viral model systems: a poxvirus (vaccinia) that infects mammalian cells; a phycodnavirus (Paramecium bursaria Chlorella virus) that infects green algae; and a baculovirus (Autographa californica nuclear polyhedrosis virus) that infects insect cells. These three viruses have large DNA genomes and they are among the most complex viruses known. Specific aims are to determine the catalytic mechanisms and define the active sites of vaccinia, baculovirus, and Chiorella virus RNA triphosphatases. These viral proteins are members of a new family of metal-dependent triphosphatases that are structurally and mechanistically distinct from the host cell RNA triphosphatase. A related aim is to determine the structure and mechanism of the vaccinia virus cap methyltransferase. These goals will be accomplished by site-directed mutagenesis, biochemical analysis, and crystallography. The proposed studies of viral capping enzymes will provide valuable insights to the evolution of a uniquely eukaryotic mRNA processing event and will also open up new approaches to antiviral drug discovery targeted at viral mRNA cap formation. Novel antipoxvirus drug targets are a pressing issue given the very real concern that smallpox might be used as a bioterror weapon. A second focus of the project involves three viral enzymes - 5' OH polynucleotide kinase, polynucleotide 3' phosphatase, and RNA ligase - that catalyze reactions implicated in RNA repair, splicing, and editing pathways. The large DNA bacteriophage T4 will be used as a model system. The long-range goals are to delineate the active sites of T4 polynucleotide kinase/phosphatase (Pnk) and T4 RNA ligase and to determine the atomic structures of these enzymes by X-ray diffraction. This component of the project will illuminate the structural basis for protein-catalyzed RNA recombination/repair events and evolutionary transitions from RNA-world to DNA-world enzymology.